MANIPULATION OF AMYLASE REACTION TO IMPROVE THE REDUCING

SUGARS PRODUCTION

CHAN CHIA SING

UNIVERSITI TEKNOLOGI MALAYSIA

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MANIPULATION OF AMYLASE REACTION TO IMPROVE THE REDUCING

SUGARS PRODUCTION

CHAN CHIA SING

A dissertation submitted in partial fulfilment of the

requirements for the award of the degree of

Master of Science (Biotechnology)

Faculty of Biosciences and Bioengineering

Universiti Teknologi Malaysia

JULY 2012

iii

To my beloved family

iv

ACKNOWLEDGEMENT

This dissertation would not have been possible without the guidance and help

of several individuals who in one way or another contributed and extended their

valuable assistance in the preparation and completion of this study. In particular, I

wish to express my sincere appreciation to my main supervisor, Dr. Goh Kian Mau,

for encouragement, guidance and critics throughout the whole research. He also gave

me the opportunity to study and providing me with the greatest stimulus for this

research topic. Without his continued support, this dissertation would not have been

the same as presented here.

Secondary, I would like to give my special appreciation to my dearest family

who support me with lots of concern and encouragement, so that I can complete this

project successfully. Their supports provide me the spirit to cope the obstacles.

Next, I would like to thank to all my friends and my laboratory colleagues for

their understanding, support and encouragements when I was facing the difficulty to

carry out the project. Special thanks to Ummirul Mukminin, Chai Yen Yen, Ranjani

Velayudhan and Chai Kian Piaw who had provided assistance at various occasions

and guided me continuously until completing this project. Their views and tips are

useful indeed.

Last but not least, I would like to thank to all the lab assistants for their

willingness to assist me and provide me with every kinds of services during the

dissertation preparation. Unfortunately, it is not possible to list all of them in this

limited space. I am grateful to all the people that I contacted with.

v

ABSTRACT

An Anoxybacillus strain SK3-4 was previously isolated from Perak Sungai

Klah hot spring. The α-amylase gene fragment from Anoxybacillus sp. denoted as

ASKA was cloned into pET-22b(+) and transformed into Escherichia coli BL21

(DE3). However, the reactivity and productivity of this amylase is underexplored.

The main objective of this project is to optimize the reducing sugars production using

Response Surface Methodology (RSM). The ASKA substrate specificity was

determined using soluble starch and nine different commercial starches: corn,

tapioca, wheat, potato, rice, sago, rye, green peas and glutinous rice starch. Sago

starch was found to be the best substrate with highest reducing sugars production.

Variable parameters such as reaction temperature, sago starch and ASKA

concentration were screened using one-factor-at-a-time (OFAT) approach before

they were optimized through two-level full factorial design and central composite

rotatable design (CCRD). Statistical analysis showed that all the three parameters

were significant factors in 23 full factorial design before further optimized the

reducing sugars production with CCRD. The final optimized parameters using

CCRD was capable to produce 7.97 g/L reducing sugars with 2.64 % (w/v) sago

starch and 0.375 unit ASKA under 66.9 ºC reaction temperature. The hydrolysis

products were determined using High Performance Liquid Chromatography (HPLC).

Maltose was the major hydrolysis product and no glucose production was detected.

As a conclusion, applying experimental designs method was able to improve the

efficiency of reducing sugars production for 87.09 % compared with the reference

reaction condition with maltose as the major end product.

vi

ABSTRAK

Satu bakteria species Anoxybacillus (SK3-4) telah berjaya dipencilkan dari

kolam air panas Sungai Klah (SK) di Perak. Species Anoxybacillus tersebut

mengandungi gen α-amilase yang di namakan sebagai ASKA. Gen α-amilase itu

telah diklonkan dalam vektor pET-22b(+) di dalam E. coli BL21 (DE3). Namun

begitu, tindak balas dan produktiviti α-amilase tersebut masih belum dikaji. Oleh

yang demikian, objektif utama kajian ini adalah untuk mengoptimumkan penghasilan

oligosakarida dengan menggunakan Response Surface Methodology (RSM).

Spesifisiti ASKA terhadap substrat telah ditentukan dengan menggunakan kanji

terlarut dan sembilan jenis kanji komersil lain iaitu kanji jagung, ubi kayu, gandum,

kentang, beras, sagu, rai, kacang hijau dan beras pulut. Kanji sagu dikenal pasti

sebagai substrat terbaik dengan penghasilan oligosakarida tertinggi. Tiga jenis faktor

iaitu suhu tindak balas, kepekatan kanji sagu dan kepekatan ASKA telah disaring

dengan menggunakan kaedah satu-faktor-pada-satu masa (OFAT). Analisis

statistikal rekabentuk 2k faktorial penuh menunjukkan bahawa ketiga-tiga faktor itu

adalah signifikan dalam mempengaruhi penghasilan oligosakarida. Ketiga-tiga faktor

itu kemudian dimanipulasi menggunakan rekabentuk komposit kebolehputaran pusat

(CCRD) untuk mengoptimumkan penghasilan oligosakarida. Keadaan tindak balas

yang optimum adalah pada suhu 66.9 ºC, 2.64 % (w/v) kanji sagu dan 0.375 unit

ASKA dengan penghasilan oligosakarida sebanyak 7.97 g/L. High Performance

Liquid Chromatography (HPLC) kemudiannya digunakan bagi menentukan produk

hidrolisis itu. Maltosa adalah produk utama hidrolisis dan tiada penghasilan glukosa

dicatat. Kesimpulannya, penggunaan rekabentuk eksperimen berjaya meningkatkan

penghasilan oligosakarida sebanyak 87.09 % daripada tindak balas rujukan dengan

maltosa sebagai produk utama hidrolisis.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xi

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xv

LIST OF APPENDICES xviii

1 INTRODUCTION 1

1.1 Background of research 1

1.2 Problem statement 3

1.3 Objectives 3

1.4 Scopes of research 3

2 LITERATURE REVIEW 4

2.1 Starch 4

2.2 Amylase 5

2.3 Alpha-amylase 6

viii

2.4 Factors influencing the hydrolysis reaction 7

2.4.1 Starch source 8

2.4.2 Starch concentration 8

2.4.3 Starch pretreatment property 9

2.4.4 Source of amylase 9

2.4.5 Enzyme concentration 10

2.4.6 Reaction temperature 10

2.4.7 pH of reaction medium 11

2.4.8 Effect of additives 11

2.5 Industrial application of amylases 12

2.5.1 Alcohol 12

2.5.2 Baking 13

2.5.3 High fructose syrup 13

2.6 Experimental design 14

2.6.1 Factorial design model (FDM) 15

2.6.2 Response surface methodology (RSM) 16

3 MATERIALS AND METHODS 17

3.1 Bacterial strain 17

3.2 Chemicals 17

3.3 General experimental design 18

3.4 Medium preparation 20

3.4.1 Luria-Bertani (LB) medium with ampicillin 20

3.4.2 Dinitrosalicylic acid (DNS) reagent 20

3.5 Bacterial stock preparation 21

3.6 α-Amylase expression and concentration 22

3.6.1 Expression of recombinant α-amylase

(ASKA)

22

3.6.2 Concentration of crude α-amylase 22

3.6.3 Amylase activity assay 23

3.7 α-Amylase substrate specificity determination 23

ix

3.8 Variable parameters screening using one-factor-at-a-

time (OFAT) approach

24

3.8.1 Starch concentration 24

3.8.2 α-Amylase concentration 24

3.8.3 Calcium chloride (CaCl2) concentration 25

3.8.4 Incubation temperature 25

3.9 Experimental design 26

3.9.1 Two-level-factorial design 26

3.9.2 Central composite rotatable design (CCRD) 28

3.9.3 Model validation 28

3.9.4 Analysis of hydrolysis products by HPLC 30

4 RESULTS AND DISCUSSION 31

4.1 α-Amylase substrate specificity determination 31

4.2 Variable parameters screening using one-factor-at-a-

time (OFAT) approach

34

4.2.1 Sago starch concentration 34

4.2.2 α-Amylase concentration 35

4.2.3 Calcium chloride (CaCl2) concentration 36

4.2.4 Reaction temperature 37

4.3 Optimization of variable parameters using Design of

Experiment

37

4.3.1 23 full factorial design 37

4.3.2 Central composite rotatable design (CCRD) 43

4.3.3 Model validation 47

4.3.4 Interaction among the variables 48

4.3.5 Model verification 54

4.4 Analysis of hydrolysis products by HPLC 55

4.4.1 Analysis of hydrolysis products in different

time interval by HPLC

56

x

5 CONCLUSION 58

5.1 Conclusion 58

5.2 Future work 59

RERERENCES 61

APPENDICES 68

xi

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 α-Amylase family members and their origin (Kuriki

and Imanaka, 1999)

7

3.1 Composition of LB medium per liter 20

3.2 Composition of 1.0 % (w/v) DNS reagent per liter 21

3.3 The actual and coded values of each parameter for 23

factorial design

27

3.4 The experimental plan for 23 factorial design in

actual and coded values. Values in the parenthesis

indicate the coded values

27

3.5 The actual and coded values of each parameter for

CCRD

28

3.6 The experimental plan for CCRD in actual and coded

values. Values in the parenthesis indicate the coded

values

29

4.1 The reducing sugars production for each substrate at

12th hour

34

4.2 The actual and coded values of each parameter for 23

factorial design

38

4.3 The experimental values and predicted values for 23

factorial design. Values in the parenthesis indicate

the coded values

41

4.4 Analysis of Variance (ANOVA) for 23 full factorial

design

42

4.5 The actual and coded values of each parameter for

CCRD

43

xii

4.6 The experimental values and predicted values for

CCRD. Values in the parenthesis indicate the coded

values

45

4.7 Analysis of Variance (ANOVA) for CCRD 46

4.8 Summary of optimum condition for each parameter

in each model design. Actual value indicates the

experimental results while predicted value indicates

the calculated response generated by the model

55

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 The structures of α-amylose and amylopectin (Stenesh,

1998)

5

3.1 The project overall experimental flow 19

4.1 The 24-hour-time plot of reducing sugars production for

ten different starches. (a) Soluble starch; (b) Tapioca

starch; (c) Potato starch; (d) Wheat starch; (e) Sago

starch; (f) Rice starch; (g) Green peas starch; (h)

Glutinous rice starch; (i) Corn starch; (j) Rye starch

32

4.2 Reducing sugars production for different sago starch

concentration

35

4.3 Reducing sugars production for different ASKA

concentration

36

4.4 Reducing sugars production for different CaCl2

concentration

36

4.5 Reducing sugars production for different reaction

temperature

37

4.6 Ramp of optimized parameters through 23 full factorial

design

40

4.7 Ramp of optimized parameters through CCRD 44

4.8 Diagnostic plots for CCRD. (a) Normal plot of residual;

(b) Plot of residuals versus predicted; (c) Outlier T plot;

(d) Box-Cox plot

48

4.9 Contour and response surface plots for the effect of sago

starch (% (w/v)) and ASKA concentration (unit)

towards reducing sugars production

51

xiv

4.10 Contour and response surface plots for the effect of sago

starch concentration (% (w/v)) and temperature (ºC)

towards reducing sugars production

52

4.11 Contour and response surface plots for the effect of

ASKA concentration (unit) and temperature (ºC)

towards reducing sugars production

53

4.12 Production of reducing sugars by various reaction

conditions

56

4.13 Production of reducing sugars at various time intervals 57

4.14 Reducing sugars production fraction at various time

intervals

57

xv

LIST OF SYMBOLS/ ABBREVIATIONS

ANOVA - Analysis of variance

ASKA - Anoxybacillus species SK3-4 alpha-amylase

B. - Bacillus

Ca2+ - calcium ion

CaCl2 - calcium chloride

CCRD - central composite rotatable design

C.I. - confidence interval

CV - coefficient of variation

DNS - 3,5-dinitrosalicylic acid

E. coli - Escherichia coli

g - gram

G1 - glucose

G2 - maltose

G3 - maltotriose

G4 - maltotetraose

G5 - maltopentaose

g/L - gram per liter

HCl - hydrochloric acid

HPLC - High Performance Liquid Chromatography

IPTG - isopropyl β-D-thiogalactopyranoside

IU - international unit

kDa - kilodalton

kPa - kilo pascal

L - liter

LB - Luria-Bertani

mg - miligram

xvi

min - minute(s)

mL - mililiter

mm - milimeter

mM - milimolar

MW - molecular weight

MWCO - molecular weight cut-off

NaCl - sodium chloride

NaOH - sodium hydroxide

nm - nanometer

OD - optical density

OD600 - optical density at 600 nm

OFAT - one-factor-at-a-time

PES - polyethersulfone

PRESS - predicted residual sum of squares

P-value - probability value

R2 - coefficient of determination

rpm - revolutions per minute

RSM - Response Surface Methodology

SK - Sungai Klah

sp. - species

Tris - tris(hydroxymethyl)methylamine

U - unit of enzyme activity

v/v - Volume per volume

w/v - weight per volume

α - alpha

µ - micro

µg - microgram

µL - microliter

µm - micrometer

µmol - micromole

% - percentage

ºC - degree Celcius

3D - three-dimensional

xvii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A List of Medium Preparation 68

B Determination of α-Amylase Activity Using DNS

Assay

69

C HPLC Standard Curves for Reducing Sugars 70

D HPLC chromatogram of various reducing sugars

standards and their retention time

73

E HPLC chromatogram of reducing sugars produced

by various reaction conditions

74

CHAPTER 1

INTRODUCTION 1.1 Background of research

Starch is one of the most abundant natural storage polysaccharides

synthesized by plants. The hydrolysis of the complex starch structure required

amylolytic enzymes to depolymerise it and form oligosaccharides and small sugars.

The world today shows an increasing interest in investigating the usage of amylolytic

enzymes for biorefinery in varieties of industries; include the food product and non-

food product industries. Amylolytic enzymes act on starch and can be categorized

into four different groups, i.e. the exo acting amylases, endo acting amylases,

debranching amylases and cyclodextrinases (Nigam and Pandey, 2009). α-Amylase

(EC 3.2.1.1) is one of the endo acting amylases (endo-1,4-α-D-glucan

glucohydrolase) which is capable to hydrolyze internal α-D-1,4-glycosidic linkages

in amylopectin and glycogen (Richardson et al., 2002).

Alpha-amylase can be found in plants, animals and microorganisms as it

plays a dominant role in their carbohydrate metabolism. Since 1980, mesophile

Bacillus licheniformis (Richardson et al., 2002) is highly used for industrial

application due to its extreme thermostability. Others α-amylase producers include B.

subtilis (Konsula and Liakopoulou-Kyriakides, 2003), B. amyloliquefaciens

(Demirkan, 2005), B. stearothermophilus (Kim et al., 1989), Aspergillus species and

Penicillium sp. (Gouda and Elbahloul, 2008). Thermophilic Anoxybacillus which

was first described by Pikuta et al. (2000) also contains the ability to undergo extra-

cellular amylase activity (Poli et al., 2006).

2

Amylases have been applied in varieties of industries; include food, textile,

paper, pharmaceutical and detergent industries (Shigechi et al., 2004). High demand

of amylases has encouraged the discovery of new amylases from different

microorganisms sources with an aim to find alternative that could lower the cost and

power requirement. Amylase reaction condition is also playing an important role for

enzyme stabilizing, which will subsequently increase the enzyme reactivity and

influence the products formation (Sivaramakrishnan et al., 2006).

An in-house Anoxybacillus strain SK3-4 was previously isolated from Sungai

Klah (Perak) hot spring. The α-amylase gene fragment from Anoxybacillus sp. was

cloned into pET-22b(+) and transformed into E. coli BL21 (DE3) (Chai, 2012). The

recombinant α-amylase (denoted as ASKA) has an optimum activity of pH 8 and 60

°C.

Physical and chemical parameters are two categories that influence the

enzymatic hydrolysis reaction (Agrawal et al., 2005). The physical parameters

include starch source, starch condition, pH of the reaction mixture, reaction

temperature and the incubation period for enzymatic reaction. While chemical

parameters are starch concentration, enzyme concentration, presence and the

concentration of divalent ions and other stabilizing agents (Richardson et al., 2002;

Sivaramakrishnan et al., 2006; Tester et al., 2006; Tamilarasan et al., 2010).

Conventional one-factor-at-a-time approach for optimization process is time

consuming and tedious. Therefore, response surface methodology (RSM) which

designs and analyzes the experimental result through mathematical and statistical

techniques can be useful to solve the complexity of one-factor-at-a-time approach

and optimize the response. In this study, two-level-full-factorial and central

composite design (CCD) will be applied to optimize the reducing sugars production

which involves various factors such as reaction temperature, starch and α-amylase

concentration.

3

1.2 Problem statement

The study of amylase from Anoxybacillus is an interesting field since the

function and reactivity of this amylase is underexplored. The application of ASKA is

an economic alternative for high temperature liquefaction process. Thus, optimize

the reducing sugars production by novel recombinant amylase is important. This

ultimately provides an alternative to produce high amount of reducing sugars with

less expenditures.

1.3 Objectives

i. To identify the best substrate for Anoxybacillus sp. amylase (ASKA).

ii. To screen the variable parameters that will influence the reducing sugars

production.

iii. To optimize the relevant factors that involve in reducing sugars production by

ASKA reaction through two-level full factorial and central composite

rotatable design (CCD).

iv. To determine the end product of ASKA hydrolysis reaction using HPLC.

1.4 Scopes of research

i. Determination of the best substrate for ASKA using nine food-grade starches.

ii. Possible reducing sugars production ranges determination using conventional

one-factor-at-a-time (OFAT).

iii. Optimization and validation of reducing sugars production by ASKA

enzymatic reaction through 23 full factorial design and central composite

design (CCD).

iv. Analysis of ASKA reaction products by HPLC.

REFERENCES Abdul-Wahab, S. A. and Abdo, J. (2007). Optimization of multistage flash

desalination process by using a two-level factorial design. Applied Thermal

Engineering. 27(2-3), 413-421.

Agrawal, M., Pradeep, S., Chandraraj, K., and Gummadi, S. N. (2005). Hydrolysis of

starch by amylase from Bacillus sp. KCA102: a statistical approach. Process

Biochemistry. 40(7), 2499-2507.

Ahmad, F. B. and Williams, P. A. (1998). Rheological properties of sago starch.

Journal of Agricultural and Food Chemistry. 46(10), 4060-4065.

Ahmad, F. B., Williams, P. A., Doublier, J., Durand, S. and Buleon, A. (1998).

Physico-chemical characterisation of sago starch. Carbohydrate Polymers.

38(4), 361-370.

Alvani, K., Qi, X., Tester, R. F. and Snape, C. E. (2010). Physico-chemical

properties of potato starches. Food Chemistry. 125(3). 958-965.

Anderson, M. J. and Whitcomb, P. J. (2005). RSM simplified: optimizing processes

using response surface methods for design of experiments. United States:

Productivity Press.

BeMiller, J. N. and Whistler, R. L. (Eds.) (2009). Starch: Chemistry and Technology.

(3rd ed.). USA: Academic Press.

Bettelheim, F. A., Brown, W. H., Campbell, M. K and Farrell, S. O. (2008).

Introduction to General, Organic and Biochemistry. (9th ed.). Canada:

Cengage Learning.

Bhunia, P. and Ghangrekar, M. M. (2008). Statistical modeling and optimization of

biomass granulation and COD removal in UASB reactors treating low

strength wastewaters. Bioresource Technology. 99(10), 4229-4238.

62

Burhan, A., Nisa, U., Gőkhan, C., Őmer, C., Ashabil, A. and Osman, G. (2003).

Enzymatic properties of a novel thermostable, thermophilic, alkaline and

chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6.

Process Biochemstry. 38(10), 1397-1403.

Chai, Y. Y. (2012). Cloning and characterisation of alpha-amylases from

Anoxybacillus sp. SK3-4 and dT 3-1. Master dissertation, University

Tecnology Malaysia, Skudai.

Chai, Y. Y., Rahman, R. N., Illias, R. M. and Goh, K. M. (2012). Cloning and

characterization of two new thermostable and alkalitolerant α-amylases from

the Anoxybacillus species that produce high levels of maltose. Journal of

Industrial Microbiology and Biotechnology. 39(5), 731-741.

Collins, B. S., Kelly, C. T., Fogarty, W. M. and Doyle, E. M. (1993). The high

maltose-producing α-amylase of the thermophilic

actinomycete,Thermomonospora curvata. Applied Microbiology and

Biotechnology. 39(1), 31-35.

Cui, R. and Oates, C. G. (1997). The effect of amylase-lipid complex formation on

enzyme susceptibility of sago starch. Food Chemistry, 65(4), 417-425.

Demirkan, E. S., Mikami, B., Adachi, M., Higasa, T. and Utsumi, S. (2005). α-

Amylase from B. amyloliquefaciens: purification, characterization, raw starch

degradation and expression in E. coli. Process Biochemistry. 40(8), 2629-

2636.

Eriksson, L. (2008). Design of Experiments: Principles and Application. (3rd ed.).

Sweden: MKS Umetrics AB.

Food and Agriculture Organization of the United Nations (2006). Starch market adds

value to cassava. Retrieved July 17, 2010, from

http://www.fao.org/ag/magazine/0610sp1.htm.

Ghorbani, D. (2008). Development of methodology for optimization and design of

chemical flooding. United States: ProQuest.

Goh, K. M., Mahadi, N. M., Hassan, O., Abdul Rahman, R. N. Z. R. and Illias, R. M.

(2007). The effects of reaction conditions on the production of γ-cyclodextrin

from tapioca starch by using a novel recombinant engineered CGTase.

Journal of Molecular Catalysis B: Enzymatic. 49(1-4), 118-126.

63

Gouda, M. and Elbahloul, Y. (2008). Statistical optimization and partial

characterization of amylases produced by halotolerant Penicillium sp.. World

Journal of Agricultural Sciences. 4(3), 359-368.

Govindasamy, S., Oates, C. G. and Wong, H. A. (1992). Characterization of changes

of sago starch components during hydrolysis by a thermostable alpha-

amylase. Carbohydrate Polymers, 18(2), 89-100.

Goyal, N., Gupta, J. K. and Soni, S. K. (2005). A novel raw starch digesting

thermostable α-amylase from Bacillus sp. I-3 and its use in the direct

hydrolysis of raw potato starch. Enzyme and Microbial Technology. 37(7),

723-734.

Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K. and Ghauhan, B. (2003).

Microbial α-amylases: a biotechnological perspective. Process Biochemistry.

38(11), 1599-1616.

Hsiu, J., Fischer, E. H. and Stein, E. A. (1963). Alpha-amylases as calcium-

metalloenzymes. II. Calcium and the catalytic activity. Biochemistry. 3(1),

61-66.

Iefuji, H., Chino, M., Kato, M. and Iimura, Y. (1996). Raw-starch-digesting and

thermostable α-amylase from the yeast Cryptococcus sp. S-2: purification,

characterization, cloning and sequencing. Biochemistry Journal. 318(3), 989-

996.

Johnston, I. A. and Bennett, A. F. (Eds.) (1996). Animals and temperature:

phenotypic and evolutionary adaptation. Great Britain: Cambridge University

Press.

Kelly, C. T., Collins, B. S, Fogarty, W. M. and Doyle, E. M (1993). Mechanisms of

action of the α-amylase of Micromonospora melanosporea. Applied

Microbiology and Biotechnology. 39(4-5), 599-603.

Kim, J., Nanmori, T. and Shinke, R. (1989). Thermostable, raw-starch-digesting

amylase from Bacillus stearothermophilus. Applied and Environmental

Microbiology. 55(6), 1638-1639.

Konsula, Z. and Liakopoulou-Kyriakides, M. (2004). Hydrolysis of starches by the

action of an α-amylase form Bacillus subtilis. Process Biochemistry. 39(11),

1745-1749.

64

Kuriki, T. and Imanaka, T. (1999). The concept of the α-amylase family: structural

similarity and common catalytic mechanism. Journal of Bioscience and

Bioengineering. 87(5), 557-565.

Lazić, Z. R. (2006). Design of Experiments in Chemical Engineering: A Practical

Guide. Germany: John Wiley and Sons.

Legin, E., Copinet, A. and Duchiron, F. (1998), A Single Step High Temperature

Hydrolysis of Wheat Starch. Starch – Stärke. 50(2-3), 84–89.

Lim, L. H., Macdonald, D. G., and Hill, G. A. (2003). Hydrolysis of starch particles

using immobilized barley [alpha]-amylase. Biochemical Engineering Journal.

13(1), 53-62.

Luan, T. (2011). Binh Dinh Export Tapioca Starch Processing JSC: Stronger Market

Foothold. Retrieved June 3, 2011, from

http://www.vccinews.com/news_detail.asp?news_id=22812.

Mamo, G. and Gessesse, A. (1999). Purification and characterization of two raw-

starch-digesting thermostable α-amylases form a thermophilic Bacillus.

Enzyme and Microbial Technology. 25(3-5), 433-438.

Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of

reducing sugar. Analytical Chemistry. 31, 426-428.

Montgomery, D. C (2005). Design and analysis of experiments. (6th ed.). USA: John

Wiley and Sons, Inc.

Mukerjea, R., Slocum, G. and Robyt, J. F. (2006). Determination of the maximum

water solubility of eight native starches and the solubility of their acidic-

methanol and –ethanol modified analogues. Carbohydrate Research. 342(1),

103-110.

Nielsen, S. S. (2010). Application of enzymes in food analysis. In Food Analysis (p.

285). New York: Springer.

Nigam, P. S. and Pandey, A. (Eds.) (2009). Biotechnology for Agro-Industrial

Residues Utilisation: Utilisation of Agro-Residues. United Kingdom:

Springer.

Pandey, A. (Ed.) (2006). Enzyme technology. Delhi: Springer.

Park, J. T. and Rollings, J. E. (1994). Effects of substrate branching characteristics

on kinetics of enzymatic depolymerization of mixed linear and branched

polysaccharides: I. Amylose/amylopectin α-amylolysis. Biotechnology and

Bioengineering. 44(7), 792-800.

65

Peatciyammal, N., Balachandar, B., Kumar, M. D., Tamilarasan, K. and

Muthukumaran, C. (2010). Statistical optimization of enzymatic hydrolysis of

potato (Solanum tuberosum) starch by immobilized α-amylase. International

Journal of Chemical and Biological Engineering. 3(3), 124-128.

Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N.,

Nikitin, D., Osipov, G. and Laurinavichius, K. (2000). Anoxybacillus

pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately

thermophilic bacterium from manure, and description of Anoxybacillus

flavithermus comb. nov.. International Journal of Systematic and

Evolutionary Microbiology. 50(6), 2109-2117.

Pishtiyski, I. and Zhekova, B. (2005). Effect of different substrates and their

preliminary treatment on cyclodextrin production. World Journal of

Microbiology and Biotechnology. 22(2), 109-114.

Poli, A., Esposito, E., Lama, L., Orlando, P., Nicolaus, G., Francesca de Appolonia,

Gambacorta, A. and Nicolaus, B. (2006). Anoxybacillus amylolyticus sp. nov.,

a thermophilic amylase producing bacterium isolated from Mount Rittmann

(Antarctica). Systematic and Applied Microbiology. 29(4), 300-307.

Reddy, T. A. (2011). Applied Data Analysis and Modeling for Engineers and

Scientists. London: Springer.

Richardson, T. H., Tan, X., Frey, G., Callen, W., Cabell, M., Lam, D., Macomber, J.,

Short, J. M., Robertson, D. E. and Miller, C. (2002). A novel, high

performance enzyme for starch liquefaction. The Journal of Biological

Chemistry. 277(29), 26501-26507.

Ryan, T. P. (2007). Modern Experimental Design. New Jersy: Wiley-Interscience.

Saboury, A. A., and Karbassi, F. (2000). Thermodynamic studies on the interaction

of calcium ions with alpha-amylase. Thermochimica Acta. 362(1-2), 121-129.

Saini, B. L. (2010). Introduction to Biotechnology. New Delhi: Laxmi Publication,

Ltd.

Schieber, A. and Saldaña, M. D. A. (2009). Potato Peels: A Source of Nutritionally

and Pharmacologically Interesting Compounds – A Review. Global Science

Books. Retrieved July 17, 2010

http://www.globalsciencebooks.info/JournalsSup/images/0906/FOOD_3(SI2)

23-29o.pdf.

66

Seager, S. L. and Slabaugh, M. R. (2010). Organic and Biochemistry for Today. (7th

ed.). USA: Cengage Learning.

Shigechi, H., Fujita, Y., Koh, J., Ueda, M., Fukuda, H. and Kondo, A. (2004).

Energy-saving direct ethanol production from low-temperature-cooked corn

starch using a cell-surface engineered yeast strain co-displaying

glucoamylase and [alpha]-amylase. Biochemical Engineering Journal. 18(2),

149-153.

Sivak, M. N. and Preiss, J. (Eds.) (1998). Starch: Basic Science to Biotechnology.

USA: Academic Press.

Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K. M., Soccol, C. R. and

Pandey, A. (2006). α-Amylases from microbial sources- an overview on

recent developments. Food technol. Biotechnol. 44(2), 173-184.

Sodhi, H. K., Sharma, K., Gupta, J. K., and Soni, S. K. (2005). Production of a

thermostable [alpha]-amylase from Bacillus sp. PS-7 by solid state

fermentation and its synergistic use in the hydrolysis of malt starch for

alcohol production. Process Biochemistry. 40(2), 525-534.

Stenesh, J. (1998). Biochemistry. New York: Birkhauser.

Swinkels, J. J. M. (1985). Composition and properties of commercial native starches.

Starch. 37(1), 1-5.

Tamilarasan, K., Ashok, R., Abinandan, S. and Kumar, M. D. (2010). Optimization

of operation variables for corn flour starch hydrolysis using immobilized α-

amylase by response surface methodology. International Journal of

Biotechnology and Biochemistry. 6(6), 841-850.

Tanaka, A. and Hoshino, E. (2003). Secondary calcium-binding parameter of

Bacillus amyloliquefaciens alpha-amylase obtained from inhibition kinetics. J

Biosci Bioeng. 99(3), 262-267.

Tester, R. F., Karkalas, J. and Qi, X. (2004). Starch-composition, fine structure and

architecture. Journal of Cereal Science. 39(2), 151-165.

Toole, A. G., Toole, G. and Toole, S. M. (2002). Essential AS Biology. United

Kingdom: Nelson Thornes.

United States Department of Agriculture (USDA) (2008). Sugar: World Production

Supply and Distribution. Retrived August, 19, 2011,

http://www.fas.usda.gov/htp/sugar/2008/Nov%20sugar%202008.pdf.

67

Van der Maarel, M. J. E. C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H.,

and Dijkhuizen, L. (2002). Properties and applications of starch-converting

enzymes of the [alpha]-amylase family. Journal of Biotechnology. 94(2),

137-155.

Voet, D. J., Voet, J. G. and Pratt, C. W. (2008). Principles of Biochemistry. (3rd ed.).

Hoboken, New Jersey: Wiley and Sons.

Wang, W. J., Powell, A. D. and Oates, C. G. (1995). Sago starch as a biomass source:

raw sago starch hydrolysis by commercial enzymes. Bioresource Technology.

55(1), 55-61.

Whitehurst, R. J. and Oort, M. V. (2009). Enzymes in Food Technology. (2nd ed.).

United Kingdom: John Wiley.

Zhang, T. and Oates, C. G. (1999). Relationship between α-amylase degradation and

physic-chemical properties of sweet potato starches. Food Chemistry. 65(2),

157-163.